Method of purifying tpa or plasminogen activator using a tripeptide of the formula: -X-Y-argininal wherein X and Y are selected from the group consisting of pro,phe,trp and tyr

ABSTRACT

A method for purifying tPA or a plasminogen activator having an active site resembling that of tPA from an impure solution thereof which comprises contacting the impure solution with a solid support having bound thereto a tripeptide of the formula: -X-Y-argininal, wherein X and Y are amino acids selected from the group consisting of pro, phe, trp and tyr. The method is also used with a tripeptide of the formula: -phe-Y-argininal, wherein Y is selected from the group consisting of phe, pro, trp, tyr, val, ile and glu(PEA).

FIELD OF THE INVENTION

This invention relates to a process for isolating plasminogen activatorsvia an affinity ligand.

BACKGROUND OF THE INVENTION

Fibrinolytic enzymes may be divided into two general classes. The firstclass of enzymes can be characterized by the ability to directly digestfibrin, and includes trypsin and plasmin. The second class indirectlydigests fibrin by activating plasminogen. The latter class, comprisingthe plasminogen activators (i.e. urokinase and tissue plasminogenactivator (tPA)), can be further characterized based on immunologicalcriteria, molecular weight and polypeptide composition (see Collen etal., Thromb Haemostas, 48:294-296 (1982)).

In general, the plasminogen activators tend to have a higher substratespecificity than trypsin or plasmin. For example, while both tPA andtrypsin recognize an arginine or lysine at the scissile bond site of asubstrate, e.g., -X-Y-Arg-↓Z-Z'-, trypsin is capable of hydrolyzing amuch broader range of peptide sequences. In fact, trypsin is capable ofautolysis. tPA by contrast appears to hydrolyze a specific peptide loopin plasminogen and does not autolyze, owing to its high specificity.

The purification of plasminogen activators, most notable tPA, has beenthe focus of extensive research in recent years. Various protocols havebeen described for the purification of tPA including: saltprecipitation, e.g. ammonium sulfate precipitation (see Rijken et al.,Biochem Biophys Acta, 580:140 (1979), Meyhack et al., EP-A-143,081); ionexchange chromatography, e.g., zinc chelate agarose (see, Rijken et al.,supra, Collen et al., U.S. Pat. No. 4,752,603, Dodd et al., FEBS,209(1):13 (1986)), SP-Sephadex® (see, Kruithof et al., J. Biochem,226:631-636 (1985)), and CM-Sepharose® (see, Murakami et al., U.S. Pat.No. 4,552,760). Another commonly used technique in conjunction with themethod(s) disclosed above is size exclusion chromatography as taught byCollen et al., U.S. Pat. No. 4,752,603, Murakami et al., U.S. Pat. No.4,552,760, and Rijken et al., Biochem Biophys Acta, 580:140 (1979).

In addition, various affinity chromatography ligands have been used inthe purification of tPA. For example, Wallen et al. (Eur J Biochem,133:681-686 (1983)) disclose the use of an anti-porcine tPA affinityligand. Meyhack et al. (EP-A-143,081) disclose anti tPA antibodies andthe Erythrina latissima trypsin inhibitor (referred to as ETI or DE-3).Dodd et al. (FEBS, 209(1):13 (1986)) report purification of tPA usinglysine as an affinity ligand. Murakami et al. (U.S. Pat. No. 4,552,760)suggest using a fibrin Sepharose® column for tPA purification. Wilson etal., (EP-A-113,319) report several purification schemes, includingaminobenzamidine Sepharose® and DE-3 Sepharose®; and Wei et al.(EP-A-178,105) disclose the use of a dye (i.e. Trisacryl blue) as anaffinity ligand.

Most of these methods are not appropriate for the large scale productionof tPA, as they are inefficient in product recovery or are onlypartially effective in removing impurities. Large scale purificationmethods which employ immunoaffinity chromatography (e.g., Wallen et al.(Eur J Biochem, 133:681-686 (1983)) and Meyhack et al. (EP-A-143,081))are limited by the cost of the antibody resin, the difficulty insterilizing this resin and by the potential for the antibodies, orfragments thereof, to leach into the recovered tPA.

Hence the need for a cost-effective affinity ligand to purifyplasminogen activators remains. In order to obtain a high degree ofpurity, a ligand with a high avidity towards a plasminogen activator isneeded. The problem then, is to identify- such ligands with a highavidity for plasminogen activators, yet without such high avidity thatthe plasminogen activator cannot be desorbed without denaturation.

SUMMARY OF THE INVENTION

The present invention relates to a method for purifying plasminogenactivators having the active site of tPA from an impure solution. Thismethod comprises contacting the impure solution with a solid supporthaving bound thereto a tripeptide of the formula -X-Y-Argininal, where Xand Y are hydrophobic amino acids and Argininal is the aldehyde analogof arginine.

This invention also relates to a solid support, having bound thereto thetripeptide of the present invention, -X-Y-Argininal, where X and Y arehydrophobic amino acids and Argininal is the aldehyde analog ofarginine.

In related aspects, this invention also comprises a plasminogenactivator bound to the tripeptide of the present invention.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to a solid support for purifyingplasminogen activating enzymes and a method for isolating saidplasminogen activating enzymes. The solid support of the inventioncomprises a tripeptide ligand with a high avidity for plasminogenactivating enzymes having the active site of tPA, herein referred to asplasminogen activators, or PA. The ligand - PA avidity must besufficiently strong so as to adsorb a PA, but must also permit thesubsequent desorption or elution without denaturation of the plasminogenactivator. In addition, this avidity must be sufficiently selective soas to not adsorb significant amounts of impurities.

The tripeptide ligands of the present invention are active-site directedtransition state analogs. These ligands are tripeptide aldehydes of thegeneral formula: -X-Y-Argininal, wherein X and Y comprise hydrophobicamino acids having non polar or polar uncharged substituents. Thecarboxy terminal peptide is an aldehyde analog of arginine, hereinreferred to as argininal or Argal.

It is preferable that the amino acid residue in the Y position behydrophobic in nature. However, it is essential that the amino acidresidue in the X position be hydrophobic. Such hydrophobic amino acidsinclude, for example, ala, val, leu, ile, pro, phe, trp, met and tyr.Preferable hydrophobic amino acids include pro, phe, trp and tyr. Otheramino acids hydrophobic in nature may include derivatives of nonhydrophobic amino acids, for example, glu(PEA) [phenethyl amido]. One ofskill in the art will appreciate that use of other amino acidderivatives, hydrophobic in nature, is also encompassed by the presentinvention. In addition, the present invention is not limited to aminoacid residues that form a peptide bond (i.e., --CONH--) between the Xand Y positions of the tripeptide ligand. For example, the X and Yresidues may form an isosteric linkage. Examples of peptide isosteresare --CH═CH--, --CH₂ CH₂ --, --COCH₂ --, --COO--, --CHOHCH₂ --, --CH₂S-- and --NHCO--.

The present invention is not intended to be bound by a particularmechanism of action. However, it is hypothesized that upon associationwith active site of a PA, the aldehyde moiety of the tripeptidecovalently reacts with the catalytic serine alcohol group to form ahemiacetal. This hemiacetal mimics the transition state of the normal PAsubstrate cleavage reaction (amidolysis) resulting in a covalently boundcomplex. Hence, variations in the tripeptide ligand may affect theselectivity and/or avidity of the ligand - PA interaction. For example,replacing the carboxy terminal peptide with the alcohol analog ofarginine (i.e., Argol) results in a less tightly formed ligand - PAcomplex without appreciable change in tripeptide selectivity. On theother hand, variations in the X and Y positions of the tripeptide ligandcan affect selectivity towards a particular PA.

For example, the tripeptide sequence Pro⁵⁵⁸ - Gly⁵⁵⁹ -Arg⁵⁶⁰ is foundadjacent to the scissile bond or cleavage site of plasminogen, asubstrate of both tPA and urokinase (UK). Table III discloses that bothtPA and UK bind to the active-site transition state ligand,Pro-Gly-Argal. However, both are readily eluted by a low salt washindicating a low avidity to this tripeptide ligand. In contrast, theactive-site transition-state ligand for a different tripeptide not foundadjacent to the scissile bond, Phe-Pro-Argal (Table IV), shows a muchhigher ligand - PA avidity. Hence there is little, if any, predictivevalue of naturally occurring substrate sequences as a basis forselecting a particular tripeptide ligand.

The tripeptide aldehyde ligand can be attached to the solid support by astepwise manner coupling of amino acid residues. Alternatively, oneskilled in the art could presynthesize the complete tripeptide aldehydeand attach it in one step to the solid support. The tripeptide aldehydesynthesis on the solid support is from the N-terminus to C-terminus,which is opposite in direction to the commonly used Merrifieldsolid-phase peptide synthesis. The methods of peptide synthesis,however, are well known in the art, see for example, Ali et al., J MedChem, 29:984 (1986) and J Med Chem, 30:2291 (1987).

To introduce argininal at the C terminus, the precursor argininesemicarbazone can first be generated by the method of Patel et al.,Biochem Biophys Res Comm, 104:181-186 (1982), which is incorporated byreference herein.

Tripeptides as described herein, are useful in affinity purification ofPAs from impure solutions. The impure solution can be, e.g., a clarifiedfermentation broth or conditioned media or any partially purifiedsolution (i.e., 0.1%-95% PA). Unlike chemically unreactive affinityligands, one which contains an argininal moiety has the potential tonon-selectively react with primary and secondary amines in buffers orculture media (e.g., amino acids, peptides, etc.). Therefore, the impuresolution containing the plasminogen activator to be purified ispreferably at a pH of 8.5 or less. More preferably, the solutioncontaining the plasminogen activator to be purified is at pH 6.0 orlower.

The solid support or solid carrier to which the ligand is coupled can beany material which is insoluble in the buffers or impure solutionsemployed and which is capable of chemically coupling the ligand. Manysuch carriers are known. These include glass, silica, alumina, andzirconia as well as organic carriers such as agarose, cellulose,dextran, polyamide, polyacrylamide and vinyl copolymers of bifunctionalacrylates with various hydroxylated monomers. Commercially availablecarriers include, for example, Affi-Gel®, Sephadex®, Sepharose®,Trisacryl®, Sepharose®, and Biogel®.

An advantage of the high avidity ligands described herein is asimplified purification scheme. This method is also expected tofacilitate lower costs of purification than currently available methods,e.g. monoclonal antibodies (mAb), fibrin column chromatography,benzamidine agarose followed by several other chromatographicprocedures.

Another advantage of the present purification process is that when usedin conjunction with a metal chelate affinity capture step and sizeexclusion chromatography, a protein with purity greater than 95% can beobtained which is designed to meet certain regulatory requirements for apharmaceutical product (e.g., clearance of DNA, viral kill steps, etc.).

Yet another advantage of the present invention includes a greaterlatitude for adsorbing the plasminogen activator. For example, a pHrange of 3.0 to 8.5, preferably 3.5 to 6.0 can be used. Furthermore, nospecific desalting step is required after the desorbing operation. tPAfor example, can be eluted and recovered in a high yield from theadsorbent by merely lowering the pH. This invention, however, is notlimited to the purification of tPA, but includes other plasminogenactivators which possess the same, or substantially the same, activesite as tPA. As used herein, the term "active site of tPA" refers toplasminogen activators capable of adsorbing to transition statetripeptides, as described herein, which remain bound during high ionicstrength wash conditions (e.g., 1.0M salt at neutral pH), yet can bedesorbed by a change in pH (e.g., elution by 0.1M acetic acid). Inaddition the "active site of tPA" refers to serine protease activity,which can be determined chromogenically, e.g., the S-2251 assay(Weinberg et al., J Immunol Meth, 75:289 (1984)) or by alternatemethods, for example, clot lysis activity as described byGranelli-Piperno et al. (J Exp Med, 148:223 (1978)). Thus plasminogenactivators having the same, or substantially the same, active site oftPA have a high avidity for transition state tripeptides of the presentinvention and also possess serine protease activity, as defined above,which is substantially the same, if not greater, than the serineprotease activity of wild type tPA.

Plasminogen activators include, for example, tPA and variants thereof,which possess the same or substantially the same active site of tPA suchas variants in which one or more amino acids have been added, deleted,rearranged, or substituted. Such variants also encompass molecules inwhich one or more functional domains have been added, deleted or alteredsuch as by combining the active site of one plasminogen activator, e.g.,tPA, with the fibrin binding domain of another plasminogen activator,e.g., one or more kringle regions from urokinase or plasminogen,.or withanother fibrin binding molecule such as a Fab fragment of an anti-fibrinIgG molecule (see for example, Runge et al., Proc Natl Acad Sci, 84:7659 7662 (1987).

Other variants include tPA molecules in which the primary amino acidsequence has been altered in the growth factor domain so as to increasethe serum half-life of the plasminogen activator. Such tPA growth factorvariants are disclosed for example, by Browne, EP-A-0,240,334 (PublishedOct. 7, 1987), Kalyan et al., W088/05822 (Published, Aug. 11, 1988), andCassani et al., EP-A-0,308,716 (Published, Mar. 29, 1989; urokinasevariants).

Preferred plasminogen activator variants include the substitutionmutations BBNT5 and BBNT12, both disclosed in G.B. patent applicationserial number GB 8815135.2, filed Jun. 24, 1988. BBNT5 consists of twosubstitution mutations in wild type tPA, Tyr⁶⁷ to Ser and Phe⁶⁸ to Ser.BBNT12 embodies 3 substitutions, Leu⁶⁶ to Asp, Tyr⁶⁷ to Asp, and Phe⁶⁸to Thr.

Further examples of preferred plasminogen activators, purified by thepresent invention, are hybrids comprising the active site of tPA, e.g.the B chain of tPA, with other sequences, e.g., the A-chain of plasmin,see Robinson et al., U.S. Pat. No. 4,752,581. Another preferred variantis disclosed by Browne et al., EP-A-297,882 (Published Jan. 4, 1989).This molecule, referred to as H37, is a plasminogen (amino acids1-541) - tPA (amino acids 262-527) hybrid and referred to herein as mut222. Other variants include fusions of the active site of tPA to otherplasminogen activators, e.g. urokinase, pro-urokinase, streptokinase.

Yet further examples of plasminogen activators having the active site oftPA are glycosylation mutants of tPA such as disclosed, for example, byWei et al., EP-A-0,178,105 (Published, Apr. 16, 1986), Haigwood et al.,EP-A-0,227,462 (Published, Jul. 1, 1987), Meyhack et al., EP-A-0,225,286(Published, Jun. 10, 1987), and Baltimore et al., EP-A-0,299,706(Published, Jan. 18, 1989).

Although it is not required, it is preferable to employ an initialcapture step which results in isolation of most of the plasminogenactivator while substantially reducing the volume of the impure solutionfor further purification. This initial capture step relies on a solidsupport capable of handling a large volume of impure solution (e.g.culture media or any other solution that contains a PA either impure orin a partially purified state, e.g. 0.1-95% PA), at a high flow rate andwith a high efficiency. Examples of such chromatography include, forexample, immobilized ligands such as metal chelating agents, lysine,arginine, fibrin, ETI, various dyes (e.g. Trisacryl Blue), boronic acid,phenylboronate, concanavalin A, p-aminobenzamidine and benzamidine. Thepreferred capture step is metal chelate chromatography, especially zincchelate chromatography. Such a technique is generally described byPorath et al., Nature, 258:598 (1975). The support can be, e.g., a softgel such as dextran, agarose or polyacrylamide gel, a rigid gel such asFractogel® vinyl polymer gel, 32-63 mm size (Pierce Chemical Co.,Rockford, Ill.) or any other support capable of adsorbing PA.

As disclosed by DePhillips et al., U.S. patent application Ser. No.07/056,927, filed Jun. 3, 1987, and incorporated by reference herewith,the purification by the zinc chelate column can be enhanced by thewashing of the adsorption support by water below pH 5.0 in an aqueoussolution of an alkali or alkaline earth metal salt e.g., NaCl, KCl,MgCl₂, CaCl₂ or a chaotrope, e.g., guanidine, thiocyanate, or urea.

It has been found, for example, that washing a zinc chelate column atabout neutral pH in accordance with the standard procedure does notremove a significant amount of contaminants from the conditioned medium.A second wash at pH less than 5.0 unexpectedly removes a significantamount of further contaminants. For example, in a representativeexperiment in which a zinc chelate column was washed completely withwater buffered to pH 6.0 and then with water buffered to pH 4.5, thefirst wash removed contaminants approximately equal to the amount of tPAand the second wash removed an unexpectedly large amount of contaminantsequal to about 10% of the amount of tPA as determined by comparison ofpeaks in a chromatogram showing absorbance at 280 nm.

The impure solution obtained from the capture step is now partiallypurified such that the tPA (or other plasminogen activators) is at least50% pure and preferably 75 to 95% pure, based on total protein contentby reverse phase chromatography, SDS-PAGE, or any other standard meansto quantitate proteins.

Prior to absorption to the tripeptide ligand, the eluant from abovecontaining the plasminogen activator may be further concentrated byselective precipitation, for example, ammonium sulfate ppt.,immunoprecipitation, and isoelectric precipitation. The impure solutionis next adsorbed onto a tripeptide affinity support. Both the D- and theL-enantiomers were equally effective as tripeptide ligands, however theD-enantiomers are preferable due to a greater resistance to proteolyticcleavage.

The affinity support is washed at neutral pH with a low salt, andsubsequently a high salt (e.g., 1.0M) wash. The salt is then removed byadditional low salt washes prior to elution. The selectively boundplasminogen activator may then be desorbed with an eluant of pH lessthan 6.0 or greater than 8.0 and may or may not contain a strongnucleophile like hydroxylamine or semicarbazide. Preferably theplasminogen activator is eluted with 0.1-1.0M acetic acid; morepreferably with 0.2-0.4M acetic acid.

Following the tripeptide column adsorption/elution, one more step may bedesirable to produce a plasminogen activator product of pharmaceuticalgrade. This can be, for example, ion exchange chromatography or sizeexclusion chromatography. Preferably, it is size exclusionchromatography.

The size exclusion step is carried out using a small particle packing,e.g., less than about 50 μm, having a separation range of 1000 to300,000 molecular weight. By the use of such gel, residual traces ofhigh molecular weight contaminants can be removed. For example,Superose® 12 cross linked agarose (Pharmacia, Piscataway, N.J.), whichhas an average particle size of 20-40 μm and a separation range of 1,000to 300,000 molecular weight, has been discovered to readily separatetPA, (molecular weight of about 70,000 Daltons), from high molecularweight (greater than 90,000 Daltons) contaminants. Such separation ofphysically similar proteins by size exclusion chromatography removesresidual PA aggregates, including dimers, nucleic acids and, if present,virus particles.

Fractions from the size exclusion step which contain the plasminogenactivator are collected for final formulation, sterile filtration andpackaging. Prior to final formulation, salt can be removed by, e.g.dialysis, diafiltration, or a desalting column, for example, Sephadex®G-25, Biogel® P2 or Biogel® P4.

The examples which follow are illustrative, but not limiting of thepresent invention.

EXAMPLES

Amino acids and protected derivatives were purchased from BachemBioscience Inc. (Philadelphia, Pa.). Affi-Gel-10 was obtained fromBio-Rad Labs (Richmond, Calif.). Chromogenic enzyme substrates werepurchased from Helena Labs (Texas). Bovine thrombin was from MilesDiagnostics (Kankakee, Ill.). Urokinase was obtained from Calbiochem(San Diego, Calif.). tPA was obtained from in-house sources, it is alsoavailable commercially (e.g. American Diagnostic, New York, N.Y.). Otherspecialty chemicals were procured from Sigma Chemical Co. (St. Louis,Mo.). Arginine semicarbazone was synthesized according to Patel et al.,Biochem Biophys Res Comm, 104:181-186 (1982).

Example 1. Synthesis of E-SEP-EDA-SA

1.0 liter of Sepharose Cl-6B (Pharmacia Fine Chemicals, catalog #17 048001) was reacted with 52.5 ml epibromohydrin in a basic solution of 0.5NNaOH/30% tetrahydrofuran (THF) at 40° C. for four hours. The activatedsepharose was collected by filtration on a sintered glass funnel and waswashed extensively with 30% THF to remove unreacted epibromohydrin. Theproduct was further washed with water until the washing was neutral.This gel was then resuspended in 1.0 liter of water and was allowed toreact with ethylenediamine (50 ml) overnight at room temperature. Thegel was filtered and unreacted ethylenediamine was removed by washingthe gel with 0.1M acetic acid followed by water. The gel was resuspendedin 1.0 liter of water, then was reacted with succinic anhydride (25 gm)at pH 6.0. The gel was further washed with 1.0 liter of a sodiumcarbonate solution (0.2M) followed by water until the washing was foundto be neutral. This gel was finally washed with isopropyl alcohol andstored at 4° C. for further use as a moist powder. This gel is hereinreferred to as E-SEP-EDA-SA.

Example 2. Synthesis of E-SEP-EDA-SA-X-Y-Argal

The "X", "Y" and ArgSC (semicarbazide protecting group) groups werecoupled sequentially to the succinyl moiety using a water-solublecarbodiimide [N ethyl N'-(3-dimethyl-aminopropyl)-carbodiimidehydrochloride] (or EDAC), triethylamine (TEA), isopropyl alcohol (IPA),and N hydroxybenzotriazole (HOBT). The X and Y groups were added asmethyl ester derivatives and followed by saponification with 0.1M sodiumcarbonate as described by Bogdanszky (Principles of Peptide Synthesis,Chap. 3, Springer-Verlag, Berlin (1984)). The ArgSC was added asdescribed by Patel et al., supra. The semicarbazide protecting group wasremoved by treatment with formaldehyde in dilute acetic acid. Theschematic of this synthesis is shown in Table I.

                  TABLE I                                                         ______________________________________                                        Synthesis of ESEPEDASAXYArgal                                                 ESEPEDASA                                                                      ##STR1##                                                                      ##STR2##                                                                      ##STR3##                                                                      ##STR4##                                                                      ##STR5##                                                                      ##STR6##                                                                     ______________________________________                                    

Example 3. Binding of Plasminogen Activators to Affinity Adsorbents

Affinity supports containing a variety of tripeptide sequences weresynthesized as described above and were tested for PA binding. In atypical binding experiment, 1 to 40 μg of plasminogen activator (PA) in10 ml of 0.1M NaOAc containing 0 to 1M NaCl at pH 4.5 was passed throughan affinity column (5ml) at a rate of 1 ml/min. The column was thenwashed with 0.1M sodium phosphate buffer at pH 7.0 followed byadditional washes of the same buffer with increased ionic strength byaddition of salt to a final concentration of 1.0M. The selectively boundenzyme was eluted with 0.1M acetic acid at 0.3 ml/min.

Samples were assayed with appropriate chromogenic substrates at roomtemperature. Absorbance changes were recorded in a spectrophotometer for300 secs. tPA samples (50 μl) were added to a solution containing 0.1MTris/0.01% PEG-3400, pH 8.1 (900 μl) and 50 μl 0.01MD-Ile-Pro-Arg-pNA.HCl (or S-2288); also assayed was substrate S-2251(Kabi Group, Greenwich, Conn.) by the method of Conners et al. (DNA,7:651 661 (1988)). Where appropriate, samples were diluted so thatlinear absorbance tracings were obtained during the enzyme reactions.

The tPA concentration was determined using various standard assaysincluding HPLC, the S2251 Assay, the particle fluorescence immunoassay(PCFIA) performed according to the protocol provided by the manufacturer(Pandex Laboratories Inc., Mundelein, Ill.), and the enzyme immunoassay(EIA) performed substantially as described in the protocol provided bythe manufacturer (American Diagnostica, New York, N.Y.).

For trypsin activity determination, trypsin samples (100 μl) were addedto 900 μl of Tris buffer (see tPA) and 100 μl of 0.01M tosylargininemethyl ester in water; absorbance was measured at 247 nm. Urokinasesamples (200 μl) were added to 800 μl of Tris buffer (see tPA) and 50 μlof 0.003M pyr-Glu-Gly-Arg-pNA.HCl (S-2444); absorbance was measured at405 nm. Thrombin samples (50 μl) were added to 900 ml of Tris buffer(0.1M Tris/0.25M CaCl₂ /0.01% PEG-3400 pH 8.1) and 50 μl of 0.002MD-Phe-Pip-Arg-pNA (S-2238); absorbance was measured at 405 nm.

Table II discloses a tripeptide ligand with a greater selective affinityfor trypsin than myeloma-derived tPA. Trypsin and tPA are known torecognize the tripeptide sequence Arg-Leu-Arg of ETI (erythrina trypsininhibitor). Hence the transition state analog, Arg-Leu-Argal, wassynthesized for binding studies. As shown in Table II, tPA initiallyabsorbed to this ligand, but was readily desorbed. In contrast, trypsinabsorbed this ligand with a much higher avidity, such that only 10% ofthe affinity bound enzyme could be released.

                  TABLE II                                                        ______________________________________                                        Binding to Affi-10-Arg--Leu--Argal                                                             % Activity                                                                    Trypsin                                                                              tPA                                                   ______________________________________                                        load               100%     100%                                              flow through       2        0                                                 0.2M NaCl/buffer wash                                                                            2        49                                                1.0M NaCl/buffer wash                                                                            2        53                                                0.2N NH.sub.4 OH elution                                                                         10       8                                                 ______________________________________                                    

In naturally occurring plasminogen, the tripeptide sequence Pro-Gly-Argis found adjacent to the scissile bond. In searching for a more avidtripeptide, the transition state ligand Pro-Gly-Argal was synthesized.Table III shows that both tPA and urokinase (UK) initially bound to theaffinity ligand. tPA was readily desorbed with buffer, while a smallerbut significant amount of UK required low pH to allow release of theenzyme.

                  TABLE III                                                       ______________________________________                                        Binding to EDA*--SA--Pro--Gly--Argal                                                          % Activity                                                                    tPA   UK                                                      ______________________________________                                        load              100%    100%                                                flow through      1       0                                                   0.1M buffer wash  81      48                                                  1M NaCl/ buffer   7       9                                                   0.1M Acetic acid  6       21                                                  ______________________________________                                         EDA* = E--SEP--EDA                                                       

Table IV shows that the tripeptide Phe-Pro-Argal is fairly effective incapturing and purifying tPA. Quite unexpected, however, was a differencein ligand affinity depending upon the chain content of the tPA.Preparations where the cell line and/or culture conditions yieldedprimarily single-chain tPA (e.g., myeloma-derived) shared a weakerligand binding than predominantly two-chain tPA (e.g., CHO-derived).

                  TABLE IV                                                        ______________________________________                                        Binding to EDA*--SA--D--Phe--Pro--Argal                                                      % Activity                                                                    tPA*  tPA**                                                    ______________________________________                                        load             100%    100%                                                 flow thru        1       0                                                    low salt         33      4                                                    high salt        2       1                                                    0.1M HOAc        74      84                                                   ______________________________________                                         Assay with S2288.                                                             *One Chain.                                                                   **Two chain.                                                             

Various tripeptide ligands were synthesized with hydrophobic residues inthe X and Y positions. Table V shows those ligands which were screenedand found not effective in tPA binding.

                  TABLE V                                                         ______________________________________                                        Binding of Myeloma tPA to Affinity Adsorbents                                                 % Enzyme Ativity                                              Ligand            Unbnd    Wash    Recvd*                                     ______________________________________                                        D--Phe--L--Ala--Argal                                                                           0        24      54                                         L--lle--L--Ala--Argal                                                                           0        73       3                                         L--lle--D--Phe--Argal                                                                           0        98       8                                         L--Val--D--Phe--Argal                                                                           0        92      10                                         L--Ala--D--Phe--Argal                                                                           0        64      34                                         L--Tyr--L--Ala--Argal                                                                           1        55      31                                         L--Tyr--L--Val--Argal                                                                           0        87      25                                         L--Gln--L--Gly--Argal                                                                           0        98       5                                         L--Pro--L--Gly--Argal                                                                           1        88       7                                         L--Glu(PEA)--D--Phe--Argal                                                                      0        42      34                                         ______________________________________                                         *0.1M HOAc                                                               

Table VI lists tripeptide ligands with hydrophobic residues in the X andY positions which were found to be effective in binding tPA.

                  TABLE VI                                                        ______________________________________                                        Binding of Myeloma tPA to Affinity Adsorbents                                                 % Enzyme Activity                                             Ligand            Unbnd    Wash    Recvd*                                     ______________________________________                                        D--Phe--D--Phe--Argal                                                                           1        0       72                                         L--Phe--D--Phe--Argal                                                                           1        0       58                                         L--Phe--L--Phe--Argal                                                                           0        0       74                                         D--Phe--L--Phe--Argal                                                                           0        0       53                                         D--Phe--L--Trp--Argal                                                                           1        0       64                                         D--Phe--L--Val--Argal                                                                           0        3       80                                         D--Phe--L--lle--Argal                                                                           0        0       59                                         D--Phe--L--Tyr--Argal                                                                           0        0       63                                         L--Tyr--D--Phe--Argal                                                                           0        1       78                                         L--Trp--D--Phe--Argal                                                                           0        0       50                                         D--Phe--L--Glu(PEA)--Argal                                                                      0        0       72                                         ______________________________________                                         *0.1M HOAc                                                               

As noted in Table IV, there is a differential affinity between theone-chain and two-chain forms of tPA for ligand D-Phe-D-Pro-Argal.However, since both forms of tPA act similarly in vivo, it is ofinterest to isolate both equally as well in any purification scheme.Table VII show a different affinity ligand for the two forms of tPA.Despite a difference in affinity, the bulk of the tPA was recovered inthe acetic acid eluant.

                  TABLE VII                                                       ______________________________________                                        Binding of tPA to EDA*--SA--D--Phe--D--Phe--Argal                                             % Activity                                                                    tPA    tPA                                                                    (CHO)* (myeloma)**                                            ______________________________________                                        unbound            7%       1%                                                1st phosphate wash                                                                              <1       0                                                  2nd phosphate wash                                                                              0        0                                                  1st phosphate/1M NaCl wash                                                                      <1       0                                                  2nd phosphate/1M NaCl wash                                                                      0        0                                                  1st 0.1M HOAC elution                                                                           0        0                                                  2nd 0.1M HOAC elution                                                                           0        68                                                 3rd 0.1M HOAC elution                                                                           70       3                                                  4th 0.1M HOAC elution                                                                           12       --                                                 ______________________________________                                         *Predominantly two chain.                                                     **Predominantly one chain.                                               

Further studies of the ligand D-Phe-d-Phe-Argal demonstrated a veryselective protease affinity. See Table VIII.

                  TABLE VIII                                                      ______________________________________                                        Binding of Proteases                                                          to EDA*--SA--D--Phe--D--Phe--Argal                                                     % Activity                                                                    tPA (Myeloma)                                                                           Thrombin    Urokinase                                      ______________________________________                                        unbound     1%          6%           1%                                       low salt wash                                                                            0           20           3                                         high salt wash                                                                           0           0           11                                         0.1M HOAC  71          4           43                                         ______________________________________                                    

Finally, in contrast to the other examples, Table IX shows a dipeptideaffinity ligand. The results summarized below imply that the Argalresidue has a significant effect on enzyme binding.

                  TABLE IX                                                        ______________________________________                                        Proteases Binding to EDA*--SA--D--Phe--D--Phe                                          tPA.sup.1                                                                           tPA.sup.2                                                                              UK.sup.3                                                                              Thrmb.sup.4                                                                          Trp.sup.5                              ______________________________________                                        unbound     0%      22%      <1%   0%      2%                                 low salt wash                                                                            1       18       36    0      72                                   high salt wash                                                                           7       11       22    1      11                                   0.1M HOAC  <1       5        9    3       8                                   ______________________________________                                         .sup.1 from CHO cells: 2chain;                                                .sup.2 from myeloma cells: 1chain;                                            .sup.3 urokinase;                                                             .sup.4 thrombin;                                                              .sup.5 trypsin.                                                          

Example 4. Purification of tPA

Recombinant tPA was expressed in a myeloma cell line in a conditionedmedium composed of 2% Bovine serum albumin (BSA), transferrin, basalmedia e.g., MEM, and Excyte at 2 mg/L (a complex lipid supplementisolated from bovine serum). Four liters of a packed cell matrix wasperfused at a rate of approximately 25 liters per day.

Chelating Sepharose® Fast Flow from Pharmacia (Piscataway, N.J.) wasused for the Metal Chelate Affinity resin, see Porath et al., Nature,258:598-599 (1975). A 3 to 6 Liter column was used in a column with a500 cm² cross sectional area. The media loading and all the wash stepswere performed at 4° C. at a flow rate of 240 cm/hr. Elution was at aslower flow rate, i.e. 120 cm/hr to keep the eluate volumes small. ZnCl₂was used to charge the resin and was added to the media at 10 ppm priorto loading. Following loading the column was washed with 5 columnvolumes of 0.1M NH₄ Ac, pH 6.0 and then with 2-3 column volumes of 0.1MNH₄ Ac/0.1M NaCl, pH 4.5. Elution was effected by 0.1M NH Ac/0.5M NaCl,pH 4.5. The column capacity for the crude tPA was approximately 2 gramsper liter of resin.

The vast majority of media components pass through the column withoutbeing retained, while the tPA is quantitatively retained. The wash at pH6.0 removes a large percentage of the residual BSA. The pH is thenlowered to 4.5 in the presence of 100 mM NaCl to remove a heterogeneouspopulation of proteins. tPA will begin to elute if more than threecolumn volumes of this buffer are applied to the column. The tPA is theneluted at 500 mM NaCl, pH 4.5. These elution conditions are distinctlydifferent from those originally worked out for tPA (Rijken et al., JBiol Chem, 256:1035-1041 (1981)), which operated at pH 7.0 and affectedelution with a histidine gradient. Fractions containing tPA asdetermined by the S-2251 assay (Weinberg et al., J Immunol Meth, 75:289(1984)) were pulled for further processing.

tPA was then concentrated by isoelectric precipitation. The zinc chelateeluate was adjusted to pH 7.0 with 1.0N NaOH and the mixture was stirredfor 30 min at 4° C. The precipitate was then collected by centrifugationat 5,000×g. The pellet was then resuspended by the addition of 0.1Macetic acid. Following solubilization the material was adjusted to 300mM NaCl with 600 mM NaCl/0.1M NH₄ Ac, pH 4.0.

This protein was then dissolved in 0.1M acetic acid/0.3M NaCl, pH 4.5and applied to a 420 ml (5×20 cm) tripeptide affinity adsorbent columnpre equilibrated with 0.1M sodium acetate buffer, pH 4.5. The resinselected for the purification of myeloma tPA was D-Phe-D-Phe-Argal (seeExample 3). The column was then washed with 0.1M sodium phosphate bufferat pH 7.0 followed by additional washes of the same buffer withincreased ionic strength by the addition of NaCl to a finalconcentration of 1.0M. This wash serves to flush out any serum albumincontaminant. This was followed by a dilute pH 7.0 sodium phosphatebuffer to wash out the NaCl. The selectively bound enzyme was elutedwith 0.1M acetic acid. Yields were typically 95% for this step, andprotein purity routinely exceeded 95%.

As a final step, the eluate from the tripeptide affinity adsorbentcolumn was concentrated on a tangential flow apparatus using 10 kDnominal molecular weight cutoff membranes. Sodium chloride was added tobring the salt concentration to 200 mM. The eluate was then applied to aSuperose® 12 column equilibrated with 200 mM NaCl and 100 mM HOAc atroom temperature. A peak migrating near the void of the column wasdetermined to be tPA by SDS-PAGE under reducing conditions. The tail ofthe product was not pooled with the rest of the material as it containslow molecular weight contaminants. Each step results in a high percentrecovery and the use of only three chromatograph steps results in atotal recovery of approximately 76%.

The above description and examples fully disclose the inventionincluding preferred embodiments thereof. However, it is appreciated thatthe invention is not limited to the particular embodiments describedabove. Modifications of the methods described above that are obvious tothose of ordinary skill in the art are intended to be within the scopeof the following claims.

What is claimed is:
 1. A method for purifying tPA or a plasminogenactivator having an active site resembling that of tPA from an impuresolution thereof which comprises contacting the impure solution with asolid support having bound thereto a tripeptide of the formula:

    -X-Y-argininal

wherein X and Y are amino acids selected from the group consisting ofpro, phe, trp and tyr.
 2. The method of claim 1 wherein X and Y areselected from the group consisting of pro, phe, and tyr.
 3. The methodof claim 1 wherein the tripeptide is selected from the group consistingof -phe-pro-argininal, -phe-phe-argininal and -phe-tyr-argininal.
 4. Themethod of claim 1 wherein the tripeptide is -D-phe-D-phe-argininal. 5.The method of claim 1 wherein the tripeptide is covalently bound to thesolid support.
 6. The method of claim 1 wherein the tripeptide is boundto the solid support by a peptide bond.
 7. The method of claim 1 whereinthe pH of the impure solution is 3.0 to 8.5.
 8. The method of claim 1which further comprises eluting the plasminogen activator bound to thetripeptide with an eluant of pH less than 6.0.
 9. The eluant of claim 10which has a pH of less than or equal to 4.5.
 10. The method of claim 1which first comprises an initial capture step of the impure solution.11. The method of claim 10 wherein the initial capture step comprisescontacting the impure solution with a metal chelate affinity support,and subsequently eluting the plasminogen activator from said affinitysupport prior to contact with the tripeptide support.
 12. A method forpurifying tPA or a plasminogen activator having an active siteresembling that of tPA from an impure solution thereof which comprisescontacting the impure solution with a solid support having bound theretoa tripeptide of the formula:

    -phe-Y-argininal

wherein Y is an amino acid selected from the group consisting of phe,pro, trp, tyr, val, ile and glu(PEA).
 13. The method of claim 12 whereinY is selected from the group consisting of val, ile and glu(PEA).